Project Summary Pneumonic plague is the deadliest form of disease caused by Yersinia pestis. Y. pestis colonizes the lung by suppressing and evading early innate immune responses. This is done in part using a type 3 secretion system (T3SS) to inject mammalian cells with a repertoire of effector proteins aimed at limiting immune cell responsiveness. Traditionally, a mouse model of infection has been used to understand the details of the progression of pneumonic plague. Though important, there are known differences in the roles of key mediators of pathogenesis between murine and human hosts. Further, the inability to extrapolate findings in animal models to human disease is an area of concern, as the failure to translate many biological findings in human trials has placed emphasis on reconsidering the utility of various of animal models. Thus, there is a need to examine infectious disease models in human tissue platforms. In the work proposed here, we seek to employ a donor human lung platform to study early host/pathogen interactions during pneumonic plague. Fully virulent strains of Y. pestis will be used to infect precision-cut human lung slices in order to identify key target cell types, and to evaluate the response of human cells and tissue to Y. pestis infection. The specific aims of the proposal are as follows: Aim 1. Evaluate Y. pestis type III secretion in human lung tissue. In Specific Aim 1, we seek to identify host cell targets of Y. pestis type III secretion in human tissue during infection. To this end, precision-cut human lung slices will be infected with Y. pestis expressing a Yop-?-lactamase hybrid protein, the translocation of which can be identified after incubation of infected cells with the fluorescent ?-lactamase substrate CCF2- AM. Target cells undergoing translocation will be identified and levels of cell death will be quantified. We predict that Y. pestis initially targets a specific population of cells in the lung for the evasion or subversion of host immunity. Importantly, this aim will answer fundamental questions regarding the initial stages of infection by identifying target host cells in a human tissue. Aim 2. Evaluate host response to Y. pestis infection. Though the anti-inflammatory capacity of the Y. pestis Yops is well-established, the host responses to Yop intoxication have not been evaluated in vivo or in the context of pulmonary infection. Importantly, the immediate inflammatory consequences, or lack thereof, have not been evaluated in humans. In specific Aim 2, we will evaluate the immediate inflammatory consequences of Y. pestis pulmonary infection in human precision cut lung slices after infection with wild-type Y. pestis or Y. pestis strains lacking each of the effector Yop proteins. This aim will give crucial insight into how the human host responds to targeting by Y. pestis type III secretion. Importantly, the data we obtain can also be used for comparative analysis to our current murine infection model.